Abstract
In mammalian cells the Golgi apparatus undergoes an extensive disassembly process at the onset of mitosis that is believed to facilitate equal partitioning of this organelle into the two daughter cells. However, the underlying mechanisms for this fragmentation process are so far unclear. Here we have investigated the role of the ADP-ribosylation factor-1 (ARF1) in this process to determine whether Golgi fragmentation in mitosis is mediated by vesicle budding. ARF1 is a small GTPase that is required for COPI vesicle formation from the Golgi membranes. Treatment of Golgi membranes with mitotic cytosol or with purified coatomer together with wild type ARF1 or its constitutive active form, but not the inactive mutant, converted the Golgi membranes into COPI vesicles. ARF1-depleted mitotic cytosol failed to fragment Golgi membranes. ARF1 is associated with Golgi vesicles generated in vitro and with vesicles in mitotic cells. In addition, microinjection of constitutive active ARF1 did not affect mitotic Golgi fragmentation or cell progression through mitosis. Our results show that ARF1 is active during mitosis and that this activity is required for mitotic Golgi fragmentation.
Highlights
The Golgi apparatus is a membrane-bound organelle that serves as a central conduit for protein and lipid modification, processing, trafficking, and secretion in all eukaryotic cells
Our results show that ADP-ribosylation factor-1 (ARF1) is active during mitosis and that this activity is required for mitotic Golgi fragmentation
Quantitation of the results showed that about 43% of membranes were vesiculated in the presence of wild type (WT) ARF1, a significant increase compared with treatment with coatomer alone (Fig. 1I)
Summary
Reagents—All reagents were from Sigma, Roche Diagnostics, or Calbiochem, unless otherwise stated. Golgi membranes (200 g) were mixed with 10 mg of IC or MC or with purified coatomer (100 g), recombinant myristoylated ARF1 (50 g), 1 mM GTP, and an ATP-regenerating system (10 mM creatine phosphate, 1 mM ATP, 20 g/ml creatine kinase, 20 ng/ml cytochalasin B), in assay buffer (50 mM Tris-HCl, pH 7.4, 0.2 M sucrose, 50 mM KCl, 20 mM -glycerophosphate, 15 mM EGTA, 10 mM MgCl2, 2 mM ATP, 1 mM GTP, 1 mM glutathione, and protease inhibitors), in a final volume of 1000 l. Fractions were diluted 3-fold with a buffer without sucrose, and membranes in each fraction were pelleted by centrifugation in a Beckman TLA55 rotor at 55,000 rpm for 60 min followed by Western blotting analysis. Images were captured with an Orca-285 camera (Hamamatsu) and the software package Openlab 4.02 (Improvision)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
